Synthesis and antitumor activity of combretastatin D-4

Article abstract of DOI:10.3987/COM-07-11221

Combretastatin D-4 1 was synthesized using electrochemical dimerization of phenols as the key step. Anodic oxidation of bromochloro-phenol 7 provided the corresponding diaryl ether 8. Manipulation of the two hydroxyl groups differenciated, provided the se

Full text of DOI:10.3987/COM-07-11221

HETEROCYCLES, Vol. 75, No. 2, 2008
291
HETEROCYCLES, Vol. 75, No. 2, 2008, pp. 291 - 296. © The Japan Institute of Heterocyclic Chemistry
Received, 14th September, 2007, Accepted, 17th October, 2007, Published online, 23rd October, 2007. COM-07-11221
SYNTHESIS AND ANTITUMOR ACTIVITY OF COMBRETASTATIN
D-4
Kanako Uno,a Takamasa Tanabe,a Takahisa Ogamino,a Ryoko Okada,b
Masaya Imoto,b and Shigeru Nishiyamaa,*
aDepartment of Chemistry, Faculty of Science and Technology, Keio University,
Hiyoshi 3-14-1, Kohoku-ku, Yokohama 223-8522, Japan bDepartment of
Biosciences and Informatics, Faculty of Science and Technology, Keio University,
Hiyoshi 3-14-1, Kohoku-ku, Yokohama 223-8522, Japan
E-mail: nisiyama@chem.keio.ac.jp
Abstract – Combretastatin D-4 1 was synthesized using electrochemical
dimerization of phenols as the key step. Anodic oxidation of bromochloro-phenol
7 provided the corresponding diaryl ether 8. Manipulation of the two hydroxyl
groups differenciated, provided the seco-acid, which on Mitsunobu reaction gave
the target molecule. Its biological assay showed new potent inhibitory activity
against cellular proliferation of human HT-29 colon carcinoma cells.
A family of cyclic diarylheptanoids, such as galeon,1 pterocarine,2 and combretastatins,3 exhibit a wide
range of biological activity, such as antitumor, antibacterial, and antiplasmodial activities. Combined
with these attractive activities, their structures often have a relatively simple structure, making them
promising leads to new drugs. A number of synthetic approaches to these natural products, have been
reported. Among them, construction of the diaryl ether4 has generally been attained by the Ullmann and
SNAr protocols, and total synthesis of galeon and pterocarine was recently accomplished by the former.5
Against such back ground, we have independently developed the phenolic oxidation methodology by
thallium(III) salts.6
O
O
O
O
HO
combretastatin D-4 1
HO
galeon
HO
O
O
O
MeO
pterocarine
HO
Figure 1. Natural diarylheptanoids

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HETEROCYCLES, Vol. 75, No. 2, 2008
Table 1. Anodic oxidation of the phenols 2 and 7
Concent.
Products/yielfds (%)
Entries Substrates
Solvents
F/mol
3
4
5
6
8
9
10
11
mM
10
10
10
10
50
10
7
10
10
10
2
1
2
2
2
2
2
2
2
7
7
7
7
7
7
7
MeNO2
MeOH
3
3
3
2
2
2
2
2
1.5
1.5
1.5
1.5
1.8
20
10
54
90
4
3
4
5
6
7
8
9
CF3CH2OH
CF3CH2OH*1
CF3CH2OH*1
CF3CH2OH*1
MeOH*1
34
25
38
11
26
15
21
14
41
34
20
61
56
44
57
6
10
6
8
2
MeOH*2
10
70
CF3CH2OH
CF3CH2OH*1
CF3CH2OH*1
CF3CH2OH*1
CF3CH2OH*3
10
11
12
13
20
20
Anode: Pt-net. Cathode: Pt-wire. Supporting salts: LiClO4 0.1 M and n-Bu4NBF4 0.1M for entry 1.
*1Addition of aq. NaOH. *2Addition of HClO4. *3Addition of n-Bu4NOH.
OR
X
O
O
X
OH
Br
OH
OH
HO
X
X
O
Br
HO
Br
Br
Br
5: R = OMe or OH
O
O
4: X = Br
9: X = Cl
2: X = Br
7: X = Cl
3: X = Br
8: X = Cl
OMe
OMe
Cl
O
3
HO
Cl
X
OH
OH
O
X
O
Br
O
Br
O
11
6: X = Br
10: X = Cl
As part of our ongoing investigation, we attempted synthesis of galeon, pterocarine, and related
compounds employing thallium(III)-mediated cyclization.7 In their biological assessment, several
synthetic intermediates showed more potent inhibitory activity against cellular proliferation of human
HT-29 colon carcinoma cells than those of natural products,13 although yields of the key oxidative
cyclization were relatively lower than the reported data using the Ullmann protocol, owing to influenced
by steric hindrance of the ring size and substitution patterns. To pursue an efficient synthetic
methodology of cyclic diarylheptanoids, we attempted construction of the diaryl ether by dimerization of
a phenol derivative under anodic oxidation conditions. The outstanding point of the electrochemical
reaction would require no harmful metals, and usage of a single phenol may be chemo-economically
efficient. With such investigation concept, synthesis of combretastatin D-4 1, isolated from the stem of
Getonia floribunda, was undertaken, although any biological activity has not been reported.3 However,
natural products exhibiting low or no biological activity may have a potential as a new lead carrying

HETEROCYCLES, Vol. 75, No. 2, 2008
293
highly specific activity. In anodic oxidation, one-electron oxidation of phenol derivatives carrying a free
hydroxyl group (ie 2 or 7) provided the spiro-dimer 3 or 8, in which the two hydroxyl groups was
differentiated, making it possible to convert into an appropriate precursor of the desired lactone structure
of 1. We describe herein our research process.
With a synthetic plan mentioned above, we examined the anodic oxidation of halogenated phenols (Table
1). Upon use of the dibromophenol 2,8 two-electron oxidation, occurred along with expected
one-electron oxidation, to give such by-products, as 4-6, along with the desired 3 (entries 2-6). Among
parameters of the oxidation reactions, solvents along with basic or acidic additives, rather than
concentration and electricity, were effective to the reaction outcome. In contrast to MeNO2 (entry 1),
MeOH and CF3CH2OH solvents under neutral conditions, provided considerable amounts of the spiro
compound 4, produced through a cationic intermediate (entries 2 and 3). Reasons that can be considered
for this result are: bromine substituents caused too low oxidation potential, which conducted unexpected
two-electron oxidation process.9 When the halogen substituents were changed to a Br-Cl pair, oxidation
of bromochloro-phenol 710 (entries 7 - 13) effected selective formation of the targeted spirodimer 8,
except entries 7-9. Moreover, addition of NaOH or n-Bu4NOH as a base, modulated the oxidation
potential, producing the radical species preferentially to form the dimmer 8 (entries 10 - 13).
HO
OTBS
OR
OH
Br
CO2H
O
Cl
O
Cl
HO
a,b
Cl
c,d
BnO
e
Cl
O
O
Br
O
8
13: R = TBS
12
14: R = H
O
f
O
HO
O
1
Scheme 1.
Synthesis of combretastatin D-4 12.
Reagents and conditions: (a) TBSOTf,
2,6-lutidine/CH2Cl2, 80%. (b) Zn-AcOH/THF, 86%. (c) 10%Pd-C, HCO2NH4/EtOH, 60 °C, 86%. (d) i)
BnBr, K2CO3/DMF, quant; ii) SO3-pyr, Et3N, DMSO/CH2Cl2, 83%; iii) NaC1O2, 2-methyl-2-butene,
NaHPO4, t-BuOH, H2O, 98%. (e) i) MeI, K2CO3/DMF, 70%; ii) TBAF/THF, 87%; iii) 1M aq.
NaOH/MeOH, 95%. (f) i) PPh3, DEAD/PhMe, 71%; ii) 10% Pd-C, HCO2NH4/EtOH, 60 °C, 89%.
With the desired spiro-dimer 8 in hand, the existing primary hydroxyl group was protected as a silyl ether,
followed by reduction with Zn/AcOH, to furnish the diaryl ether 12. The halogen atoms were removed
by a hydrogen transfer method, and the phenol group was protected as a benzyl ether. The free hydroxyl

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HETEROCYCLES, Vol. 75, No. 2, 2008
group was oxidized in two steps to form carboxylic acid 13. Compound 13 was transformed to a methyl
ester temporarily during the desilylation process, and the following hydrolysis provided seco acid 14.
Mitsunobu reaction11 of 14 smoothly proceeded, and successive reductive removal of the benzyl group
produced combretastatin D-4 1, spectroscopic data of which was superimposable to the reported data.3,12
We also examined IC50 values (µg/mL) of synthetic 1, along with galeon and several synthetic
compounds carrying closely related cyclic diaryl ether structures,7 against proliferation of human HT-29
colon carcinoma cells (Fig. 2).13 It was noteworthy that combretastatin D-4 1, synthesized in this
investigation, exhibited inhibitory activity (IC50 18.4 µg/ml), although 1 has been reported to exhibit no
remarkable biological activity. Further structure - activity relationship studies of 1 and the biologically
active chlorinated derivatives are in progress.
O
O
O
O
O
HO
HO
galeon (>30)
O
O
O
combretastatin D-4 1 (18.4)
MeO
HO
(14.8)
O
Cl
O
O
Cl
Cl
O
HO
O
O
BnO
(8.14)
Br
(>30)
HO
HO
(>30)
Figure 2. Inhibitory activity (IC50 µg/mL) against proliferation of human HT-29 colon carcinoma cells
In conclusion, we have completed the synthesis of combretastatin D-4 1. In addition, biological
assessment of this compound newly showed the inhibition of cell proliferation of colon carcinoma cells.
Further investigation will be conducted to acquire promising leads to new chemotherapeutic agents,
despite relatively simple cyclic diaryl ether structures.
ACKNOWLEDGEMENTS
This work was supported by Grant-in-Aid for the 21st Century COE program “Keio Life Conjugated
Chemistry” and Scientific Research C (17510182) from the Ministry of Education, Culture, Sports,
Science, and Technology, Japan.
REFERENCES AND NOTES
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H. Kim, C.-S. Lee, M.-H. Woo, S.-H. Lee, Y. D. Jhang, and J.-K. Son, J. Nat. Prod., 2002, 65, 1707.

HETEROCYCLES, Vol. 75, No. 2, 2008
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8539; G. R. Pettit, S. B. Singh, and M. L. Niven, J. Org. Chem., 1990, 55, 2797. Combretastatin
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7. Unpublished result.
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This compound was prepared by essentially the same procedure as previously reported; see, F. Doi,
T. Ohara, T. Ogamino, K. Higashinakasu, K. Hasegawa, and S. Nishiyama, Bull. Chem. Soc. Jpn.,
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1
12. IR (film) 1504, 1518, 1597, 1730, 2925, 3422 cm-1; H-NMR δ 2.10 (2H, m), 2.25 (2H, t, J = 5.2
Hz), 2.82 (4H, m), 4.06 (2H, t, J = 4.8 Hz), 5.29 (1H, d, J = 2 Hz), 5.53 (1H, s), 6.60 (1H, d, J = 6
13
Hz), 6.93 (1H, d, J = 8 Hz), 7.01 (2H, d, J = 8.4 Hz), 7.30 (2H, d, J = 8.8 Hz); C-NMR δ 27.1,
28.7, 32.8, 34.0, 63.9, 112.5, 114.9, 121.4, 123.5, 131.0, 132.6, 137.8, 142.4, 149.0, 154.1, 173.8.
HREIMS found m/z 298.1199, calcd for C18H18O4: M, 298.1205.
13. The inhibition of human HT29 colon carcinoma cells was measured by MTT assay. The cells were
seeded at 2 x 104 cells well in 96-well plates. After overnight incubation, cells were treated for 48
h with increasing concentrations of compound (up to 30 µg/mL). Then MTT (5 mg/mL in PBS)

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was added to the cultures and incubated for another 4 h. The growth medium as removed and
DMSO was added to each well to dissolve purple crystals of formazan. The absorbance was
measured in a spectrophotometer at a wave length of 570 nm. The IC50 value was defined as the
compound concentration needed to inhibit 50% of growth relative to the untreated control.